Chemical derivatives and their application as antitelomerase agents

ABSTRACT

The present invention relates to cancer therapy and to novel anticancer agents having a mechanism of action which inhibits telomerase. It also relates to novel chemical compounds as well as their therapeutic application in humans.

This application is a division of U.S. application Ser. No. 10/040,370,filed Jan. 9, 2002, now allowed, which claims the benefit of U.S.Provisional Application No. 60/270,164, filed Feb. 22, 2001, and FrenchApplication No. 0100205, filed Jan. 9, 2001, all of which areincorporated herein by reference in their entirety.

The present invention relates to cancer therapy and to novel anticanceragents having a mechanism of action which is quite specific. It alsorelates to novel chemical compounds as well as their therapeuticapplication in humans.

The present invention relates to the use of novel non-nucleotidechemical compounds which interact with specific structures ofdeoxyribonucleic acid (DNA). These novel compounds consist of adistribution agent linked to an aminoaromatic group. These novelcompounds are useful in the treatment of cancers and typically act astelomerase-inhibiting agents. They are also useful for stabilizing DNAin G-quadruplex structures (e.g., guanine tetrads). The inhibition oftelomerase via the stabilization of these G-quadruplexes generallyresults in the termination of cellular mitosis and the death ofrapidly-dividing cells such as cancer cells. It may also result in theinduction of senescence in cancer cells. Thus, such telomeraseinhibiting agents have important therapeutic applications.

The compounds of the present invention have the advantage, from thetherapeutic point of view, of blocking telomerase. From a biologicalpoint of view, telomerase allows the addition of repetitive DNAsequences of the T T A G G G type (termed telomeric sequences) to theend of the telomere during cell division. Through this action,telomerase renders the cell immortal. Indeed, in the absence of thisenzymatic activity, the cell loses 100 to 150 bases at each division,which rapidly renders it senescent. During the development ofrapidly-dividing cancer cells, these cells were found to possesstelomeres which were maintained at a stable length during cell division.In these cancer cells, telomerase was found to be highly activated andallowed the addition of repetitive motifs of telomeric sequences at theend of the telomere. This allowed conservation of the length of thetelomeres in the cancer cells. During the past few years, more than 85%of cancer cells have tested positive for the presence of telomerase,whereas somatic cells do not show this characteristic.

Thus, telomerase is an important target for treating cancer cells. Thefirst approach for blocking telomerase was the use of nucleotidestructures (Chen et al., Proc. Natl. Acad. Sci. USA 93(7), 2635-2639).Diaminoanthraquinones (Sun et al., J. Med. Chem. 40(14), 2113-6) anddiethyloxadicarbo-cyanins (Wheelhouse R. T. et al., J. Am. Chem. Soc.120:3261-2, 1998) are among the non-nucleotide compounds which have beenused.

Patent WO 99/40087 describes the use of compounds which interact withthe G-quadruplex structures. Such G-quadruplex structures are typicallyperylene compounds and carbocyanins containing at least seven rings,including two heterocycles.

It has been discovered, quite surprisingly, that simple structures couldachieve a result which is at least equivalent with structures which area lot less complicated from a chemical point of view. The compounds ofthe present invention which meet the intended objective, i.e., whichbind the G-quadruplex structure and thereby exhibit atelomerase-inhibiting activity, correspond to the following generalformula: nitrogen-containing aromatic ring —NR₃— distribution agent—NR′₃— nonaromatic hydrocarbon chain

-   -   in which        -   the nitrogen-containing aromatic ring represents:            -   a quinoline optionally substituted with at least                -   a group N(Ra)(Rb) in which Ra and Rb, which are                    identical or different, represent hydrogen or a                    C1-C4 alkyl radical or                -   a group ORa in which Ra is as defined above            -   a quinoline possessing a nitrogen atom in quaternary                form or            -   a benzamidine or            -   a pyridine        -   R3 and R′3, which are identical or different, represent            independently of each other hyrdogen or a C1-C4 alkyl            radical        -   the distribution agent represents:            -   a triazine group optionally substituted with an alkyl                radical having 1 to 4 carbon atoms, a thio, oxy or amino                radical which are themselves optionally substituted with                one or more short-chain alkyl chains containing 1 to 4                carbon atoms or alternatively a halogen atom or            -   a carbonyl group or            -   a group C(⊚NH)—NH—C(═NH) or            -   an alkyldiyl group containing 3 to 7 carbon atoms or            -   a diazine group optionally substituted with the same                groups as triazine or one of its salts.

For the purposes of the above formula, nonaromatic hydrocarbon chain isunderstood to mean an alkyl (C1-C4) or alkenyl (C2-C4) chain, which islinear or branched, or a cycloalkyl (C3-C18), cycloalkenyl (C3-C18) orheterocycloalkyl (C3-C18) chain. The heterocycloalkyl group optionallyincludes the nitrogen atom.

It is of course understood that the nonaromatic hydrocarbon chain may beoptionally substituted with one or more atoms or radicals chosen fromamong halogen atoms, hydroxyl, aryl, heteroaryl, alkyloxy, aryloxy,thio, alkylthio, arylthio, amino, alkylamino and/or arylamino,dialkylamino, diarylamino, amidino, guanidino, alkylcarbonylamino,arylcarbonylamino, carboxyl, alkyloxycarbonyl, aryloxycarbonyl,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, alkylcarbonyl, arylcarbonyl, cyano,trifluoromethyl, and combinations thereof.

The alkyl chains of the optional substituents of the hydrocarbon chainmay contain 1 to 4 carbon atoms; and the aryl groups of the optionalsubstituents of the hydrocarbon chain may contain 5 to 18 carbon atoms.

In one embodiment, the compounds include a distribution agent chosenfrom a triazine and a diazine group. Suitable diazine groups includepyrimidines and quinazolines. The hydrocarbon chains may be alkyl chainscontaining 2 to 3 carbon atoms; and the heterocycloalkyl or cycloalkylchains may contain 4 to 7 carbon atoms.

Suitable triazines include the compounds corresponding to formula (I)below:

in which:

-   -   A represents        -   an amino group of formula NR1R2 in which R1 and R2, which            are identical or different, represent hydrogen or a straight            or branched alkyl group containing 1 to 4 carbon atoms or        -   a group OR1 or SR1 in which R1 has the same meaning as above            or        -   an alkyl group containing 1 to 4 carbon atoms or a            trifluoromethyl group or        -   a hydrogen atom or        -   a halogen atom chosen from fluorine, chlorine, bromine, and            iodine    -   R3 and R′3, which are identical or different, represent        independently of each other hydrogen or a C1-C4 alkyl radical,    -   Ar₁ represents:    -   a nitrogen-containing aromatic ring representing:        -   a quinoline optionally substituted with at least            -   a group N(Ra)(Rb) in which Ra and Rb, which are                identical or different, represent hydrogen or a C1-C4                alkyl radical or            -   a group ORa in which Ra is as defined above        -   a quinoline possessing a nitrogen atom in quaternary form or        -   a benzamidine or        -   a pyridine attached at the 4-position or fused with an aryl            or heteroaryl group optionally substituted with a C1-C4            alkyl group    -   alk represents        -   an alkyl unit containing 2 to 3 linear or branched carbon            atoms substituted with an amino, alkylamino, arylamino,            dialkylamino, diarylamino, or combination thereof        -   an alkenyl unit containing 2 to 3 carbon atoms substituted            with an amino, alkylamino, arylamino, dialkylamino,            diarylamino, or combination thereof        -   a heterocyclyl unit containing from 4 to 7 carbon atoms            or one of its salts.

It is evident that the quinoline motifs may be substituted by any othergroup not involved in the intended application; thus, acridine,isoquinoline, quinazoline, quinoxaline, phthalazine, benzothiazine,benzoxazine, phenoxazine, and phenothiazine groups are included in thedefinition of the quinoline groups.

In one embodiment, compounds of formula (I) include those comprising aheterocycle chosen from the 4-aminoquinolyl, 4-alkyl- and4-dialkyl-aminoquinolyl, 4-aminoquinolinium and quinolinium groups inwhich the quinolinium ring is optionally substituted with a methylgroup.

Group A may represent methylthio, amino, alkylamino or dialkylaminoradical, in which the alkyl groups in the radicals possess 1 to 4 carbonatoms.

The nonaromatic hydrocarbon chain may represent a 2-(dialkylamino)ethyl,3-(dialkylamino)propyl, 2-(N-alkyl-N-arylamino)ethyl, or3-(N-alkyl-N-arylamino)propyl chain in which the alkyl groups maycontain 1 to 4 carbon atoms or, in an alternative embodiment, 1 to 2carbon atoms; and the aryl groups may contain 5 to 18 carbon atoms or,in an alternative embodiment, 6 carbon atoms.

Another subject of the present invention relates to the compounds offormula (I) as novel chemical products. It therefore relates to thenovel products corresponding to the following formula (I):

in which:

-   -   A represents        -   an amino group of formula NR1R2 in which R1 and R2, which            are identical or different, represent a straight or branched            alkyl group containing 1 to 4 carbon atoms or        -   a group OR1 or SR1 in which R1 represents hydrogen or has            the same meaning as above or        -   an alkyl group containing 1 to 4 carbon atoms or a            trifluoromethyl group or        -   a hydrogen atom or        -   a halogen atom chosen from fluorine, chlorine, bromine, and            iodine    -   R₃ and R′₃, which are identical or different, represent        independently of each other a hydrogen atom or a C1-C4 alkyl        group,    -   Ar₁ represents:    -   a nitrogen-containing aromatic ring representing:        -   a quinoline optionally substituted with at least            -   a group N(Ra)(Rb) in which Ra and Rb, which are                identical or different, represent hydrogen or a C1-C4                alkyl radical or            -   a group ORa in which Ra is as defined above        -   a quinoline possessing a nitrogen atom in quaternary form or        -   a benzamidine or        -   a pyridine attached at the 4-position or fused with an aryl            or heteroaryl group optionally substituted with a C1-C4            alkyl group    -   alk represents        -   an alkyl unit containing 2 to 3 linear or branched carbon            atoms substituted with an amino, alkylamino, arylamino,            dialkylamino, diarylamino, or combination thereof        -   an alkenyl unit containing 2 to 3 carbon atoms substituted            with an amino, alkylamino, arylamino, dialkylamino,            diarylamino, or combination thereof        -   a heterocyclyl unit containing from 5 to 7 carbon atoms            or one of its salts.

In one embodiment, Ar₁ represents a group chosen from among 4-amino-,4-methylamino- and 4-dimethylamino-quinolyl and quinolinium groups, inwhich the quinolinium nucleus is optionally substituted with a methylgroup.

The A group may represent an amino or dimethylamino or methylthio group.

The compounds of general formula (I)include those for which thenonaromatic hydrocarbon chain may represent a 2-(dialkylamino)ethyl,3-(dialkylamino)propyl, 2-(N-alkyl-N-arylamino)ethyl or3-(N-alkyl-N-arylamino)propyl chain in which the alkyl groups contain 1to 4 carbon atoms, or 1 to 2 carbon atoms; and the aryl groups contain 5to 18 carbon atoms or 6 carbon atoms.

For example, the nonaromatic hydrocarbon chain may represent a2-(N-m-tolyl-N-ethylamino)ethyl chain.

Another subject of the present invention relates to the use of thecompounds of formula (I) as pharmaceutical products for human use.

The methods of preparing the compounds of formula (I)

are described below.

In the case where Ar₁ and Alk are present, the triazine of generalformula (A) may be obtained by sequential displacement of the halogenatoms, most generally of chlorine atoms, from the products of generalformula (B), by the amines Ar₁ and then Alk of general formula (C),according to scheme 1:

Generally, the procedure is carried out with 1 mole ofdihalo-s-triazine, or trihalo-s-triazine, and 1 mole of amine Ar₁. Theprocedure is typically carried out in an inert solvent, such as acetone,which is optionally aqueous; or an alcohol which is optionally aqueous,such as ethanol; or a halogenated solvent, such as dichloromethane; oran ether, such as diethyl ether or dioxane; or a polar aprotic solvent,such as DMF, DMSO or NMP. In one embodiment, the procedure is carriedout at a temperature of between 20° C. and 50° C. Next, 1 mole of amineAlk is added to the product of general formula (D), which may beoptionally isolated. The procedure is generally carried out at atemperature of between 50° C. and the reflux temperature.

It is also possible to carry out the procedure under the conditionsdescribed in J. Fluor. Chem., 1988, 39(1), 117-123, which is hereinincorporated by reference.

General Method 2

According to a second method, the products of general formula (A) inwhich Ar are as defined above and R represents a group NR1R2 or OR1 orSR1 may also be prepared by nucleophilic displacement of a halogen atom,generally a chlorine atom, from a product of general formula (A) inwhich R represents a halogen atom. This second method is performedaccording to scheme 2:

The procedure is generally carried out by condensing 1 mole of productof general formula (A) in which R represents a halogen atom, preferablya chlorine atom, with 1 mole of amine R1R2NH or alcoholate R1O⁻ orthioalcoholate R1S. The reaction takes place in an inert medium underthe reaction conditions. There may be mentioned among the inert solventsacetone, which is optionally aqueous; or an alcohol, which is optionallyaqueous such as ethanol; or a halogenated solvent, such asdichloromethane; or an ether, such as diethyl ether or dioxane; or apolar aprotic solvent, such as DMF, DMSO or NMP. When the entering groupis a R1R2NH group, the procedure is typically carried out at atemperature of between 20° C. and the reflux temperature, in thepresence of an organic base such as triethylamine, or an inorganic basesuch as sodium hydroxide or sodium or potassium carbonate. It is alsopossible not to use a base during the amination reaction, and to isolatea hydrochloride of the product of general formula (A), the base of whichcan then be released. When the entering group represents a R1O⁻ or R1S⁻group, the procedure is typically carried out with an alkali metal oralkaline-earth metal alcoholate or thioalcoholate, such as a sodium orpotassium or lithium or ammonium or cesium or barium salt, in a polaraprotic solvent such as DMF or DMSO or NMP, at a temperature of between50° C. and the reflux temperature.

General Method 3

According to a third method of preparing the compounds, for which Rrepresents a hydrogen atom or a straight or branched alkyl groupcontaining from 1 to 4 carbon atoms, the compounds may be prepared bycondensation of a bisguanide of general formula (E), with an acidderivative, such as an acid chloride or a methyl ester of generalformula (F) according to scheme 3:

The condensation between the bisguanide of general formula (E) and theacid derivative of general formula (F) is generally carried out in analcohol such as methanol or ethanol. The procedure is typically carriedout at a temperature of between 0° C. and the reflux temperature.

The symmetric or asymmetric bisguanides of general formula (E) may beobtained by carrying out the procedure under the conditions described inthe literature, for example, according to Patent J.P. 94-4993.

General Method 4

It is understood that the s-triazines, in general, may be obtained inthe form of libraries, by applying the methods described in schemes 1,2, or 3 in parallel and/or combinatorial chemistry in liquid phase or insolid phase. It is generally understood that when the work is carriedout in solid phase, any one of the reagents may be attached beforehandonto a solid support, chosen according to the chemical reactioninvolved, and that such a chemical reaction is followed by an operationof cleaving the product of the reaction from the solid support.

The present invention also relates to therapeutic compositionscontaining a compound according to the invention, in combination with apharmaceutically acceptable carrier. Such a carrier is typically chosenin accordance with the desired mode of administration. Thepharmaceutical composition may be provided in solid, liquid or liposomeform.

Suitable solid compositions include powders, gelatin capsules, andtablets. Among the oral forms, it is also possible to provide solidforms which are protected from the acidic medium of the stomach. Thecarriers used for the solid forms may comprise inorganic carriers suchas phosphates, carbonates, or organic carriers such as lactose,celluloses, starch or polymers. The liquid forms may comprise solutions,suspensions or dispersions. They may also contain, as a dispersivecarrier, either water, or an organic solvent (ethanol, NMP and thelike), or mixtures of surfactants and solvents, or mixtures ofcomplexing agents and solvents.

The administered dose of the compounds of the invention will be adjustedby the practitioner according to the route of administration to thepatient and the condition of the patient.

The compounds of the present invention may be administered alone ormixed with other anticancer agents. Suitable agents include, but are notlimited to:

-   -   alkylating agents such as cyclophosphamide, melphalan,        ifosfamide, chlorambucil, busulfan, thiotepa, prednimustine,        carmustine, lomustine, semustine, steptozotocin, decarbazine,        temozolomide, procarbazine and hexamethylmelamine    -   platinum derivatives such as cisplatin, carboplatin or        oxaliplatin    -   antibiotic agents such as bleomycin, mitomycin, dactinomycin,    -   antimicrotubule agents such as vinblastine, vincristine,        vindesine, vinorelbine, taxoids (paclitaxel and docetaxel)    -   anthracyclines such as doxorubicin, daunorubicin, idarubicin,        epirubicin, mitoxantrone, losoxantrone    -   group I and II topoisomerases such as etoposide, teniposide,        amsacrine, irinotecan, topotecan and tomudex,    -   fluoropyrimidines such as 5-fluorouracil, UFT, floxuridine,    -   cytidine analogs such as 5-azacytidine, cytarabine, gemcitabine,        6-mercaptomurine, 6-thioguanine    -   adenosine analogs such as pentostatin, cytarabine or fludarabine        phosphate    -   methotrexate and folinic acid    -   enzymes and various compounds such as L-asparaginase,        hydroxyurea, trans-retinoic acid, suramine, dexrazoxane,        amifostine, herceptin as well as estrogenic and androgenic        hormones.

It is also possible to combine a radiation treatment with the compoundsof the present invention. These treatments may be administeredsimultaneously, separately or sequentially. The treatment is typicallyadapted by the practitioner to the patient to be treated.

The G-quadruplex stabilizing activity may be determined by a methodusing the formation of a complex with fluorescein, as described below.

Oligonucleotides

All the oligonucleotides, modified or otherwise, were synthesized byEurogentec SA, Seraing, Belgium. The oligonucleotide FAM+DABCYL carriesthe catalog reference OL-0371-0802. It has the sequence:GGGTTAGGGTTAGGGTTAGGG (SEQ ID NO:l) corresponding to 3.5 repeats of thehuman telomeric motif (strand rich in G). The fluorescein is chemicallyattached to the 5′ end, and the DABCYL to the 3′ end, as described byEurogentec. The concentration of the samples is checked byspectrophotometry, recording the absorbance spectrum between 220 and 700nm and using the molar extinction coefficient provided by the supplier.

Buffers

All the experiments were carried out in a 10 mM sodium cacodylate bufferpH 7.6 containing 0.1 M Lithium Chloride (or Sodium Chloride). Theabsence of fluorescent contamination in the buffer was checkedbeforehand. The fluorescent oligonucleotide is added at the finalconcentration of 0.2 μM.

Study of Fluorescence

All the measurements of fluorescence were carried out on a SpexFluorolog DM1B apparatus, using an excitation line width of 1.8 nm andan emission line width of 4.5 nm. The samples are placed in amicroquartz cuvette of 0.2×1 cm. The temperature of the sample iscontrolled by an external water bath. The oligonucleotide alone wasanalyzed at 20, 30, 40, 50, 60, 70 and 80° C. The emission spectra arerecorded using an excitation wavelength of 470 nm. The excitationspectra are recorded using either 515 nm or 588 nm as emissionwavelength. The spectra are corrected for the response of the instrumentby reference curves. A high extinction (80-90%) of the fluorescence offluorescein at room temperature is observed, in agreement with anintramolecular folding of the oligonucleotide at 20° C in the form of aG-quadruplex. Such folding induces juxtaposition of the 5′ and 3′ endsof the oligonucleotide, which are respectively linked to fluorescein andto DABCYL. This juxtaposition causes an already-described phenomenon ofextinction of fluorescence which is used for “Molecular Beacons”.

Fluorescence Tm:

An oligonucleotide stock solution at the strand concentration of 0.2 μMin 0.1 M LiCl, 10 mM cacodylate buffer, pH 7.6, is prepared beforehand,heated briefly at 90° C. and slowly cooled to 20° C., and thendistributed in aliquots of 600 μl in the fluorescence cuvettes. Three μlof water (for the control) or 3 μl of test product (stock at 200 μM,final concentration 1 μM) are then added and mixed. The samples are thenallowed to incubate for at least 1 hour at 20° C. before eachmeasurement. The use of longer incubation times (up to 24 hours) has noinfluence on the result obtained.

Each experiment allows the measurement of only one sample. The latter isfirst incubated at an initial temperature of 20° C., heated to 80° C.over 38 minutes, left for 5 minutes at 80° C. and then cooled to 20° C.over 62 minutes. During this time, the fluorescence is measuredsimultaneously at two emission wavelengths (515 nm and 588 nm) using 470nm as the excitation wavelength. A measurement is carried out every 30seconds. The temperature of the water bath is recorded in parallel. Thefluorescence profile as a function of the temperature is reconstitutedfrom these values. The fluorescence profiles are then normalized between20° C. and 80° C. The temperature for which the intensity of emission at515 nm is the mean of those at high and low temperature is called theTm. Under these conditions, the Tm of the reference sample withoutaddition of product is 44° C. in a Lithium Chloride buffer. Thistemperature is increased to more than 55° C. in a Sodium Chloridebuffer. The addition of a G-quadruplex stabilizing compound induces anincrease in the Tm. This increase is judged to be significant if it isgreater than 30.

The antitelomerase biological activity is determined by the followingexperimental protocol:

Preparation of the Extract Enriched in Human Telomerase Ectivity:

The leukemia line HL60 is obtained from ATCC (American Type CultureCollection, Rockville USA). The cells are cultured in suspension in RPMI1640 medium containing L-Glutamine at 2 mM, Penicillin 200 U/ml,streptomycin 200 μg/ml, gentamycin 50 μg/ml and supplemented with 10%heat-inactivated fetal calf serum.

An aliquot of 10⁵ cells is centrifuged at 3000×G and the supernatantdiscarded. The cell pellet is resuspended by several successivepipettings in 200 μl of lysis buffer containing 0.5% CHAPS, 10 mMTris-HCl pH 7.5, 1 mM MgCl₂, 1 mM EGTA, 5 mM β-mercaptoethanol, 0.1 mMPMSF and 10% glycerol and is stored in ice for 30 minutes. The lysate iscentrifuged at 16 0000×G for 20 minutes at 4° C., and 160 μl ofsupernatant is recovered. The proteins in the extract are assayed by theBradford method. The extract is stored at −80° C.

Assay of the Telomerase Activity:

The inhibition of the telomerase activity is determined by a protocolfor extension of the oligonucleotide TS (^(5′)AATCGTTCGAGCAGAGTT^(3′))(SEQ ID NO:2), in the presence of a cellular extract enriched intelomerase activity and compounds which are added at variousconcentrations (10, 1, 0.1 and 0.01 μg/ml). The extension reaction isfollowed by a PCR amplification of the extension products with the aidof the oligonucleotides TS and CXext(^(5′)GTGCCCTTACCCTTACCCTTACCCTAA^(3′)). (SEQ ID NO:3)

The reaction medium is prepared based on the following composition: TrisHCl pH 8.3  20 mM MgCl2  1.5 mM Tween 20 0.005% (W/V) EGTA  1 mM DATP 50 μM DGTP  50 μM DCTP  50 μM DTTP  50 μM Oligonucleotide TS  2 μg/mlOligonucleotide CXext  2 μg/ml Bovine serum albumin  0.1 mg/ml Tag DNApolymerase  1 U/ml alpha 32P dCTP (3000 Ci/mmol)  0.5 μl Telomeraseextract 200 ng in a volume of 10 μl Test product or solvent in a volumeof 5 μl Double distilled water QS  50 μl

The oligonucleotides are obtained from Eurogentec (Belgium) and arestored at −20° C. at a stock concentration of 1 mg/ml in distilledwater.

The reaction samples are assembled in 0.2 ml PCR tubes and one drop ofparaffin oil is deposited on each of the reactions of the experimentbefore closing the tubes.

The reaction samples are then incubated in a Cetus 4800-type PCRapparatus under the following temperature conditions:

-   -   15 minutes at 30° C.,    -   1 minute at 90° C.,    -   followed by 30 cycle of,    -   30 seconds at 94° C.,    -   30 seconds at 50° C.,    -   and 1 minute 30 seconds at 72° C.,    -   followed by a final cycle of 2 minutes at 72° C.

For each of the samples, an aliquot of 10 μl is pipetted under the oillayer and mixed with 5 μl of a loading buffer containing: TBE 3Xglycerol   32% (W/V) Bromophenol blue 0.03% Xylene cyanol 0.03%

The samples are then analyzed by electrophoresis on 12% acrylamide gelin a 1×TBE buffer for 1 hour at a voltage of 200 volts, with the aid ofa Novex electrophoresis system.

The acrylamide gels are then dried on a sheet of whatmann 3MM paper at80° C. for 1 hour.

The analysis and the quantification of the reaction products are carriedout with the aid of an InstantImager apparatus (Pacard).

For each compound concentration tested, the results are expressed aspercentage inhibition of the reaction and calculated from the untreatedenzymatic control and from the enzyme-free sample (blank) according tothe following formula:(Compound Value−blank value/enzymatic control value−blank value)×100.

The concentration of compound inducing a 50% inhibition of thetelomerase reaction (IC50) is determined with the aid of asemilogarithmic graphical representation of the inhibition valuesobtained as a function of each of the compound concentrations tested.

A compound is considered to be active as an antitelomerase agent whenthe quantity inhibiting 50% of the telomerase reaction is less than 5μM.

The Cytotoxic Biological Activity on Human Tumor Lines is DeterminedAccording to the Following Experimental Protocol:

The human cell lines KB and A549 are obtained from ATCC (American TypeCulture Collection, Rockville USA). The A549 cells are cultured in alayer in a culture flask in RPMI 1640 medium containing L-Glutamine at 2mM, Penicillin 200 U/ml, streptomycin 200 μg/ml and supplemented with10% heat-inactivated fetal calf serum. The KB cells are cultured in alayer in a culture flask in Dulbelco's medium containing L-Glutamine at2 mM, Penicillin 200 U/ml, streptomycin 200 μg/ml and supplemented with10% heat-inactivated fetal calf serum.

The cells at the exponential growth phase are trypsinized, washed in 1XPBS and are inoculated in 96-well microplates (Costar) in an amount of4×10⁴ cells/ml for A549 and of 1.5×10⁴ cells/ml (0.2 ml/well) and thenincubated for 96 hours in the presence of variable concentrations ofproduct to be studied (10, 1, 0.1 and 0.01 μg/ml, each point inquadruplicate). 16 hours before the end of the incubation, 0.02% finalof neutral red is added to each well. At the end of the incubation, thecells are washed with 1×PBS and lysed with 1% sodium lauryl sulfate. Thecellular incorporation of the dye, which reflects cellular growth, isevaluated by spectrophotometry at a wavelength of 540 nm for each samplewith the aid of a Dynatech MR5000 reading apparatus.

For each compound concentration tested, the results are expressed aspercentage inhibition of cellular growth and calculated from theuntreated control and the culture medium free of cells (blank) accordingto the following formula:(Compound Value−blank value/cell control value−blank value)×100.

The concentration of compound inducing a 50% inhibition of growth (IC50)is determined with the aid of a semilogarithmic graphical representationof the inhibition values obtained as a function of each of the compoundconcentrations tested.

A compound is considered to be active as cytotoxic agent if theconcentration inhibiting the growth of the tumor cells tested by 50% isin particular less than 10 μM.

The following and nonlimiting examples are given to illustrate theinvention.

EXAMPLE 1 Parallel Synthesis of Substituted Derivatives ofN6-[6-amino-4-methylsulfanyl-[1,3,5]triazin-2-yl]-2-methylquinoline-4,6-diamine

Preparation ofN6-(6-chloro-4-methylsulfanyl-[1,3,5]triazin-2-yl)-2-methylquinoline-4,6-diamine

4.4 g (25 mmol) of 2-methylquinoline-4,6-diamine (which may be preparedaccording to J. Med. Chem., 35:252, 1992) and 2.8 g (25 mmol) of sodiumcarbonate are successively added, in a 1 liter three-necked flask, to asolution of 5 g (25 mmol) of2,6-dichloro-6-methylsulfanyl-[1,3,5]triazine (which may be preparedaccording to J. Amer. Chem. Soc., 67:662, 1945), in 400 ml oftetrahydrofuran. The reaction mixture is heated under reflux for 16hours. After evaporation of the tetrahydrofuran, the residue is taken upin 400 ml of a mixture of water and dichloromethane (50-50 by volume).The organic phase is separated after settling out, dried over sodiumsulfate and concentrated to dryness under reduced pressure. 7.5 g (88%)ofN6-(6-chloro-4-methyl-sulfanyltriazin-2-yl)-2-methylquinoline-4,6-diamineare then obtained, in the form of a pale yellow solid whosecharacteristics are the following:

melting point=294° C.

¹H NMR spectrum (300 MHz, (CD₃)₂SO d6, δ in ppm): 2.43 (s: 3H); 2.52 (s:3H); 6.47 (s: 1H); 6.61 (unresolved complex: 2H); 7.62 (broad d, J=9 Hz:1H); 7.69 (d, J=9 Hz: 1H); 8.32 (unresolved complex: 1H); 10.80(unresolved complex: 1H).

Parallel Synthesis ofN6-[6-(2-dimethylaminoethylamino)-4-methylsulfanyl-[1,3,5]triazin-2-yl]-2-methylquinoline-4,6-diamine(Example 1-1)

50 mg (0.15 mmol) ofN6-(6-amino-4-methyl-sulfanyl-[1,3,5]triazin-2-yl)-2-methylquinoline-4,6-diamineare introduced into a heating magnetic reactor with a Zymark condenser,of the STEM RS2050 type, containing 25 wells in parallel each providedwith a 50 ml glass tube. 5 ml of dioxane, 16 mg (0.15 mmol) of sodiumcarbonate, 23 mg (0.15 mmol) of sodium iodide and 27 mg (0.3 mmol) of2-dimethylaminoethylamine are successively added to the first tube(Example 1-1). The reaction medium is heated by reflux and under argonfor 24 hours. After cooling, the content of the tube is evaporated underreduced pressure, taken up in 5 ml of water and 5 ml of ethyl acetateand filtered. The organic phase is separated by settling out, dried andconcentrated under reduced pressure. The crude product obtained is thenpurified by LC/MS using a Waters Xterra 3.5 μM C18 silica column 3 mm indiameter and 50 mm in length, eluting with a linear elution gradientconsisting, at the starting time (t₀=0 min), of water containing 0.05%trifluoroacetic acid and, at the final time (t_(f)=4 min), ofacetonitrile containing 0.05% trifluoroacetic acid. 58 mg ofN6-[(6-(methylquinolin-6-ylamino)-4-methylthiotriazin-2-yl]quinaldine-4,6-diaminetrifluoroacetate are thus obtained, after purification, whosecharacteristics are the following:

-   -   mass spectrum (DAD-TIC)=454 (MH⁺)    -   retention time=2.69 min (the retention times are obtained on a        hypersil C 18 5 μm column 50 mm diameter 4.6 mm trade mark        Purity Elite, eluting with a mixture of solvents A (H20/TFA        0.05%) and B (ACN/TFA 0.05%) with a linear gradient ranging from        95% A/5% B (t=0 min) to 10% A/90% B at t=3.5 min, then step 2        min).

Examples 1-1 to 1-26 were obtained by carrying out the procedure asabove in a Zymark STEM RS2050 reactor. The structures, the variousoperating conditions used and the characteristics of Examples 1-1 to1-26 are summarized in the table below: Example Structure Reactionconditions Characteristics No.of mmol Mass Retention AlkN(R 3- SolventHeating of amine MH⁺ time (min) 1-1

dioxane 17 h./100° 0.3 384 2.69 1-2

dioxane 17 h./100° 0.3 410 2.91 1-3

dioxane 17 h./100° 0.15 411 2.86 1-4

dioxane 3d/100° 0.45 422 2.85 1-5

dioxane 17 h./100° 0.15 396 2.84 1-6

dioxane 17 h./100° 0.15 424 2.79 1-7

dioxane 17 h./100° 0.15 410 2.72 1-8

dioxane 2d./100° 0.3 452 2.81 1-9

dioxane 10 ml/DMF1% 24 h./100° 0.3 425 2.43 1-10

dioxane 10 ml/DMF1% 24 h./100° 0.3 410 2.51 1-11

dioxane 10 ml/DMF1% 24 h./100° 0.3 424 2.50 1-12

dioxane 10 ml/DMF1% 24 h./100° 0.3 398 2.46 1-13

dioxane 10 ml/DMF1% 24 h./100° 0.3 398 2.48 1-14

dioxane 10 ml/DMF1% 24 h./100° 0.3 412 2.47 1-15

dioxane 10 ml/DMF1% 24 h./100° 0.3 384 2.48 1-16

dioxane 10 ml/DMF1% 24 h./100° 0.3 396 2.49 1-17

dioxane 10 ml/DMF1% 24 h./100° 0.3 411 2.38 1-18

dioxane 10 ml/DMF1% 24 h./100° 0.3 459 2.62 1-19

dioxane 10 ml/DMF1% 24 h./100° 0.3 410 2.44 1-20

dioxane 10 ml/DMF1% 24 h./100° 0.3 410 2.52 1-21

dioxane 10 ml/DMF1% 24 h./100° 0.3 422 2.55 1-22

dioxane 10 ml/DMF1% 24 h./100° 0.3 400 2.36 1-23

dioxane 10 ml/DMF1% 24 h./100° 0.3 384 2.40 1-24

dioxane 10 ml/DMF1% 24 h./100° 0.3 440 2.58 1-25

dioxane 10 ml/DMF1% 24 h./100° 0.3 410 2.48 1-26

dioxane 10 ml/DMF1% 24 h./100° 0.3 474 2.86

EXAMPLE 2 Parallel Synthesis of Substituted Derivatives ofN6-[6-amino-4-diethylamino-[1,3,5]triazin-2-yl]-2-methyl-quinoline-4,6-diamine

Preparation ofN6-(6-chloro-4-diethylamino-[1,3,5]triazin-2-yl)-2-methylquinoline-4,6-diamine

3.91 g (22.5 mmol) of 2-methylquinoline-4,6-diamine (which may beprepared according to J. Med. Chem. 35:252, 1992), and 2.4 g (22.5 mmol)of sodium carbonate are successively added, in a 1 liter three-neckedflask, to a solution of 5 g (22.5 mmol) of commercial2,6-dichloro-4-diethylamino-[1,3,5]triazine in 300 ml oftetrahydrofuran. The reaction mixture is heated to reflux for 20 hours.After evaporation of the tetrahydrofuran, the residue is taken up in 400ml of a mixture of water and dichloromethane (50-50 by volume). Theorganic phase is separated after settling out, dried over sodium sulfateand concentrated to dryness under reduced pressure. 7.4 g (92%) ofN6-(6-chloro-4-diethylaminotriazin-2-yl)-2-methylquinoline-4,6-diamineare thus obtained in the form of a yellow solid whose characteristicsare the following:

melting point=120° C.

¹H NMR spectrum (300 MHz, (CD₃)₂SO d6, δ in ppm): 1.14 (mt: 6H); 2.42(s: 3H); from 3.50 to 3.70 (mt: 4H); 6.47 (s and unresolved complex: 3Hin total); 7.54 (broad d, J=9 Hz: 1H); 7.67 (dd, J=9 and 2 Hz: 1H); 8.27(unresolved complex: 1H); 10.09 (unresolved complex: 1H).

Parallel Synthesis ofN6-[(6-(3-dimethylaminopropylamino)-4-diethylamino-[1,3,5]triazin-3-yl]-2-methylquinoline-4,6-diamine(Example 2-1)

50 mg (0.13 mmol) ofN6-(6-chloro-4-diethylamino-[1,3,5]triazin-2-yl)-2-methylquinoline-4,6-diamineare introduced into a heating magnetic reactor with a Zymark condenser,of the STEM RS2050 type, containing 25 wells in parallel each providedwith a 50 ml glass tube. 5 ml of DMF, 19 mg (0.14 mmol) of potassiumcarbonate, 21 mg (0.14 mmol) of sodium iodide and 14 mg (0.14 mmol) of3-dimethylaminopropylamine are successively added to the first tube(Example 2-1).

The reaction medium is heated at 120° C. under argon for 16 hours. Aftercooling, the content of the tube is evaporated under reduced pressureand taken up in 5 ml of water, filtered and washed with diethyl ether.The crude product obtained is then purified by LC/MS using a WatersXterra 3.5 μM C18 silica column 3 mm in diameter and 50 mm in length,eluting with a linear elution gradient consisting, at the starting time(t₀=0 min), of water containing 0.05% trifluoroacetic acid and, at thefinal time (t_(f)=4 min), of acetonitrile containing 0.05%trifluoroacetic acid. 12 mg ofN6-[(6-(3-dimethyl-aminopropylamino)-4-diethylamino-[1,3,5]triazin-2-yl]-2-methylquinoline-4,6-diamineare thus obtained, after purification, whose characteristics are thefollowing:

-   -   mass spectrum (DAD-TIC)=423 (MH⁺)    -   retention time=0.79 min (the retention times are obtained on a        hypersil C 18 5 μm column 50 mm diameter 4.6 mm trade mark        Purity Elite, eluting with a mixture of solvents A (H2O/TFA        0.05%) and B (ACN/TFA 0.05%) with a linear gradient ranging from        95% A/5% B (t=0 min) to 10% A/90% B at t=3.5 min, then step 2        min).

Examples 2-1 to 2-2 were obtained by carrying out the procedure as abovein a Zymark STEM RS2050 reactor. The structures, the various operatingconditions used and the characteristics of Examples 2-1 to 2-2 aresummarized in the table below: Example Structure Reaction conditionsCharacteristics No.of mmol Mass Retention AlkN(R 3- Solvent Heating ofamine MH⁺ time (min) 2-1

DMF 16 h./120° 0.14 423 0.79 2-2

DMF 16 h./120° 0.14 421 0.79

Table of biological results TRAP G-4 Cytotoxicity telomerase ΔTm A549Example IC50 μM ° C. IC50 μM 1-1 0.79 6 1-2 0.5 5.6 7.5 1-3 4.4 3.1 1-40.1 5.6 1-5 1.6 2.8 1-6 1.36 1-7 0.47 1-8 0.98 8.5 1-9 1.64 7 1-10 0.947 1-11 1.1 4.5 1-12 3.1 1-13 2.9 1-14 3.2 1-15 4.6 1-16 1.29 1-17 1.61-19 1 1-20 3.1 1-21 0.7 1-22 3.2 1-23 3.8 1-24 3.9 1-25 1.5 10 1-260.86 33 <0.3 2-1 0.90 7.9 2-2 5.4 2.4

1. A compound which binds the G-quadruplex structure of a telomerecomprising the following general formula: nitrogen-containing aromaticring —NR₃— distribution agent —NR′₃— nonaromatic hydrocarbon chainwherein 1) the nitrogen-containing aromatic ring represents: a) aquinoline optionally substituted with at least i) a group N(Ra)(Rb) inwhich Ra and Rb, which are identical or different, represent hydrogen ora C1-C4 alkyl radical or ii) a group ORa in which Ra is as defined aboveb) a quinoline possessing a nitrogen atom in quaternary form c) abenzamidine or d) a pyridine, 2) R₃ and R′₃, which are identical ordifferent, represent independently of each other, hydrogen or a C1-C4alkyl radical, 3) the distribution agent represents: a) a carbonyl groupor b) a group C(═NH)—NH—C(═NH) or c) an alkyldiyl group containing 3 to7 carbon atoms or d) a diazine group, a diazine group substituted with(i) an alkyl radical having 1 to 4 carbon atoms, (ii) a thiol radical,(iii) a hydroxy radical, or (iv) an amino radical, wherein the alkyl,thiol, hydroxy or amino radicals are unsubstituted or substituted withi) one or more short-chain alkyl groups containing 1 to 4 carbon atomsor ii) a halogen atom, or a salt thereof.
 2. The compound according toclaim 1, wherein the distribution agent is a diazine group.
 3. Thecompound according to claim 2, wherein the diazine group is pyrimidineor quinazoline.
 4. The compound according to claim 1, wherein thenonaromatic hydrocarbon chain is chosen from among i) alkyl (C1-C4),alkenyl (C2-C4), wherein the alkyl and alkenyl are linear or branched,ii) cycloalkyl (C3-C18) iii) cycloalkenyl (C3-C18) iv) heterocycloalkyl(C3-C18) and v) heterocycloalkyl (C3-C18) including the nitrogen atom ofthe NR′₃ group.
 5. The compound according to claim 4, wherein thenonaromatic hydrocarbon chain is unsubstituted or substituted with oneor more atoms or radicals chosen from among halogen atoms, hydroxyl,aryl, heteroaryl, alkyloxy, aryloxy, thiol, alkylthio,. arylthio, amino,alkylamino, arylamino, dialkylamino, diarylamino, amidino, guanidino,alkylcarbonylamino, arylcarbonylamino, carboxyl, alkyloxycarbonyl,aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, alkylcarbonyl, arylcarbonyl, cyano,trifluoromethyl, and combinations thereof.
 6. The compound according toclaim 5, wherein the alkyl chains comprise substituents having ahydrocarbon chain containing 1 to 4 carbon atoms, and the aryl groupscomprise substituents having a hydrocarbon chain containing 5 to 18carbon atoms.
 7. The compound according to claim 4, wherein the alkylchains contain 2 to 3 carbon atoms, and the heterocycloalkyl orcycloalkyl chains contain 5 to 7 carbon atoms.
 8. A therapeuticcomposition comprising a compound according to claim 1 and one or moreanticancer compounds.
 9. The composition according to claim 8, whereinthe one or more anticancer compounds are chosen from among alkylatingagents, platinum derivatives, antibiotic agents, antimicrotubule agents,anthracyclines, group I and II topoisomerases, fluoropyrimidines,cytidine analogs, adenosine analogs, L-asparaginase, hydroxyurea,trans-retinoic acid, suramine, irinotecan, topotecan, dexrazoxane,amifostine, herceptin, estrogenic hormones, and androgenic hormones. 10.A method of using the composition of claim 9, wherein the individualcompounds are administered in a therapeutically effective amount to apatient simultaneously, separately or sequentially.
 11. A method ofinhibiting telomerase activity, comprising administering atherapeutically effective amount of one or more compounds of claim 1 toa patient, wherein the level of telomerase activity in the patientfollowing the administration is reduced relative to the level oftelomerase activity existing prior to the administration.
 12. A methodof treating a cancer, comprising administering a therapeuticallyeffective amount of one or more compounds of claim 1 to a patient inneed of such a treatment, wherein the level of telomerase activityfollowing the administration is reduced relative to the level oftelomerase activity existing prior to the administration.
 13. Apharmaceutical composition comprising one or more compounds of claim 1,and a pharmaceutically acceptable carrier.
 14. A therapeutic combinationconsisting of the administration of one or more compounds according toclaim 1 and the administration of radiation.